HIV-3 retrovirus and its use

ABSTRACT

Described is a new variety of retrovirus designated HIV-3, samples of which are deposited in the European Collection of Animal Cell Cultures (ECACC) under V88060301. Further described are antigens obtained from the virus, particularly proteins p12, p16, p25 and glycoproteins gp41 and gp120 to be used in the diagnosis of ARC or AIDS caused by HIV-3. Immunogenic compositions to be used as vaccines contain an envelope glycoprotein of HIV-3 such as gp41 or gp120.

This application is a divisional of pending application Ser. No.08/486,836 filed Jun. 7, 1995, which is a divisional of Ser. No.08/228,519 filed Apr. 15, 1994 (issued as U.S. Pat. No. 5,567,603),which is a divisional of application Ser. No. 07/460,913 filed Mar. 23,1990 (issued as U.S. Pat. No. 5,304,466), which claims benefit under 35U.S.C. § 120 of PCT/EP89/00643 filed Jun. 8, 1989 and claims priorityunder 35 U.S.C. § 119 of EP 88 109 200.1 filed Jun. 9, 1988.

Substantial progress has been made in our understanding of the acquiredimmunodeficiency syndrome or AIDS. The prinicipal causative agent hasbeen demonstrated to be a non-transforming retrovirus with a tropism forT4 helper/inducer lymphocytes (1,2) and it has been estimated thatmillions of people world-wide have already been infected. Infection withthis virus leads, at least in a significant percentage of cases, to aprogressive depletion of the T4 lymphocyte population with aconcomittant increasing susceptibility to the opportunistic infectionswhich are characteristic of the disease.

Epidemiological studies indicate that human immunodeficiency virus type1 (HIV-1), the etiological agent responsible for the majority of AIDScases and which is currently the most widely disseminated HIV, probablyhad its origins in Central Africa (3). The discovery of this virus didnot necessarily imply the existence of other types of humanimmunodeficiency viruses. Nevertheless, a second group of humanimmunodeficiency-associated retroviruses, human immunodeficiency virustype 2 (HIV-2), was identified in West Africa (4,5). An HIV-2 virus isdisclosed in EP-A-0 239 425. An HIV-1 virus is disclosed in WO 86/02383.Other similar, but not identical, retroviruses have also been isolatedfrom simian sources (simian immunodeficiency virus, SIV) such as Africangreen monkeys (6,7) and macaques (8,9). The simian isolates have beenshown to be genetically more closely related to HIV-2 than HIV-1 but arenevertheless distinct (10).

One characteristic of human immunodeficiency viruses which complicatestheir comparison is their genetic variability; genetic variants arisespontaneously and with high frequency. A comparison of various HIV-1isolates revealed that some regions of the genome are highly variablewhile others are reasonably well conserved (11-16). Similarpolymorphisms have also been observed for HIV-2 (17). The regions withthe greatest genetic stability are presumably those regions coding forthe regions of viral proteins which are structurally or enzymaticallyessential. The viral genes with the greatest overall genetic stabilityare the gag and pol genes, while some regions of the env gene and thegenes coding for regulatory proteins such as art, tat, sor and 3'orfexhibit a high degree of variability. Some of the major structuralfeatures of the gag and pol gene products are apparently shared not onlyby all of the variants of a particular HIV type, but have, at least tosome extent, been conserved between virus types. Antiserum producedagainst HIV-1 crossreacts with the gag and pol gene products of HIV-2,albeit with a lower affinity than for the corresponding HIV-1 geneproducts. However, in spite of the demonstrable immunologicalcrossreaction, at the nucleic acid level there is little sequencehomology and no significant hybridization between these two viruses canbe detected except under very low stringency conditions (17).

A higher degree of relatedness can be demonstrated between SIVagm(STLV-III agm, nearly or completely identical to Human LymphotropicVirus type 4 (15)) and HIV-2. Immunological crossreaction is not limitedonly to the gag and pol gene products but extends to the env geneproducts as well. Nevertheless, genomic analysis of SIVagm and HIV-2showed them to be genetically distinguishable (19). DNA probes specificfor HIV-2, although able to hybridize to SIVagm sequences, hybridizepreferentially to HIV-2 (18).

We now report the isolation and characterization of a novel humanimmunodeficiency virus from a Camerounian woman and her partner.Geographically, this virus comes from a region in Africa located betweenWest Africa where HIV-2 is endemic, and East-Central Africa where HIV-1is endemic. This isolate is shown immunologically to be antigenicallymore closely related to HIV-1 than is HIV-2, yet an analysis of partialcleavage products obtained by chemical cleavage of the gag and pol geneproducts demonstrate that this isolate is neither HIV-1 nor HIV-2. Thisnovel isolate could represent an evolutionary link between HIV-1 andHIV-2. This novel virus will be referred to as HIV-3 hereinafter.

Subsequent to the filing of this application, the medical industry andscientific community has recognized the change in classification ofHIV-3 to HIV-1 subtype 0. See, e.g., Rayfield et al., EmergingInfectious Diseases 2:209-212 (1996); Janssens et al., AIDS 8:1012-1013(1994); Simon et al., AIDS 8:1628-1629 (1994); Gurtler et al., Journalof Virology 68:1581-1585 (1994); and Vanden Haesevelde et al., Journalof Virology 68:1586-1596 (1994).

Accordingly, the invention relates to an HIV-3 retrovirus or variants ofthis virus having the essential morphological and immunologicalproperties of the retrovirus deposited in the European Collection ofAnimal Cell Cultures (ECACC) under V 88060301.

A virus isolation was performed from blood from an asymptomaticCamerounian woman who is the partner of an HIV-seropositive man withgeneralized lymphadenopathy. Serum from the woman was moderatelypositive (ratio O.D./cut-off of 4.5) in the enzyme- linked immunosorbentassay (EIA, Organon Teknika) and had a low titer (1/40) in theimmunofluorescent antibody assay for HIV-1 but gave ambiguous results inthe HIV-1 Western blot assay with clear bands at p33, P53/55 and p64 butvery weak bands at p24, gp41 and gp120. The virus was isolated byco-cultivation of the woman's lymphocytes with PHA-stimulatedlymphocytes from healthy uninfected donors in a medium consisting ofRPMI 1640 buffered with 20 mM HEPES (hydroxyethylpiperazineethanesulfonate) and supplemented with 15% fetal calf serum, 5 g/mlhydrocortisone, 75 u/ml interleukin-2 (IL-2) and 2 g/ml polybrene.

After 52 days in culture, virus was detected in the culture as judged bythe presence of syncytia and on the basis of positive immunofluorescenceobserved when a laboratory reference anti-HIV antiserum was incubatedwith acetone-fixed cells from the culture. The presence of reversetranscriptase was also detected in the culture supernatant (10 4 cpm/ml,27×background). Cell-free culture supernatant was used to passage thevirus on fresh lymphocytes. After 15 days, CPE was again observed andreverse transcriptase detected in the supernatant. The virus was furtherpropagated in PHA-stimulated lymphocytes from healthy blood donors andwas transferred to continuous cell lines of leukemic origin.Virus-containing supernatant was tested in parallel with culturesupernatants known to contain HIV-1 in the differential antigencapturing test which is described in detail below. The results of thiscomparison indicated that the new isolate was not HIV-1.

The new virus was then characterized with respect to its proteinantigens and nucleic acids. The cell lines used for propagating thevirus can be, depending on the case, lines of the CEM, HUT, Molt-4, orMT4 type, or any other immortalized cell line which bears the T4receptor on its cell surface.

A preferred cell line for the continuous propagation of HIV-3 is Molt-4.Molt-4 cells infected with HIV-3 were deposited with the ECACC on Jun.3, 1988 under number V 88060301. Establishment of a chronically-infectedcell line can, for example, be carried out as follows:

Molt-4 cells (10 6/ml) and preferably Molt-4 clone 8 cells (obtainedfrom N. Yamamoto, Yamaguchi, Japan) are cocultured with infected humanlymphocytes (10 6/ml) in RPMI 1640 culture medium buffered with 20 mMHEPES and containing 10% fetal calf serum. Within one to two weeks, acytopathic effect is observed in the culture which is followed by celldeath. A fraction of the cells in the culture survive the infection andproduce virus continuously. With continued culturing, these cellsincrease in number and can be passaged. Supernatants from these cellscan be used a a source of virus.

Furthermore, the invention relates to a purified retrovirus having theessential morphological and immunological properties described below. Inmany cases, the unique characteristics of HIV-3 can best be appreciatedby comparison with the same type of characteristics relating to theother human immunideficiency viruses, HIV-1 and HIV-2.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIGS. 1 to 14 the designations HIV-3 (ANT 70) and HIV-3 (ANT 70 NA)refer to two strains of a new HIV-3 virus isolated from a Camerounianwoman and her partner from which HIV-3 (ANT 70) has been deposited underECACC V88060301.

FIG. 1 shows a procedure for preparing cleavage maps of viral proteins.

FIG. 2 shows differential antigen capturing on virus-containing culturesupernatants.

Differential antigen capturing is performed as described hereinafter.The solid line represents the results obtained using a broad-spectrumanti-HIV-1 IgG while the broken line depicts the results obtained usingan IgG which was rather specific for HIV-1. FIG. 2A, FIG. 2B, FIG. 2C,FIG. 2D, and FIG. 2E each shows a typical titration obtained with HIV-1.FIG. 2F shows the result obtained with HIV-3 (ANT 70) containingsupernatant.

FIG. 3A shows differential antigen capturing on HIV-1 and FIG. 3B showsdifferential antigen capturing on HIV-3 (ANT 70 NA) supernatants.

Differential antigen capturing was performed as described hereinafter.The solid line depicts the results obtained on plates coated with thebroad spectrum anti-HIV IgG while the broken line represents the resultsobtained on plates coated with IgG which shows less crossreactivity withHIV types other then HIV-1.

FIG. 4A shows the reactivity of anti-HIV sera on HIV-1 and FIG. 4B showsthe reactivity of anti-HIV sera on HIV-2 Western Blot strips.

The reactivities of 3 different sera on HIV-1 and HIV-2 Western blotstrips are shown. Sera: 1. anti-HIV-1, 2. anti-HIV-3 (ANT 70), 3.anti-HIV-2 (isolate 53). The molecular weights indicated are those givenby the manufacturer (Dupont Biotech).

FIG. 5 relates to the comparison of gag and pol proteins of severalHIV-1 isolates, HIV-2rod and HIV-3 (ANT 70).

Proteins were separated electrophoretically and blotted as describedlater. The blot was incubated with a broad-spectrum anti- HIV antiserumfollowed by (anti-human IgG)/alkaline phosphatase- labeled conjugate tovisualize the proteins.

A. HIV-2rod, B. an HIV-1 laboratory isolate, C. HIV-3 (ANT 70), D. anHIV-1 laboratory isolate, E. HIV-1 (SF4).

FIG. 6 shows a comparison of HIV-3 (ANT 70) and HIV-3 (ANT 70 NA)proteins.

Proteins were separated electrophoretically and blotted as describedlater. The blot was incubated with the BSR antiserum followed by(alkaline phosphatase)/anti-human IgG conjugate to visualize theproteins. Lane 1: HIV-3 (ANT 70 NA), lane 2: HIV-3 (ANT 70), lane 3:HIV-1 (SF4). The apparent intensity difference between lanes 1 and 2 iscaused by the difference in the amount of material loaded.

FIG. 7 relates to the ability of various human anti-HIV-1 sera tocapture viral antigens.

A number of human sera were diluted 1:1000 and coated directly onmicrowell plates. Detergent-treated culture supernatants containingHIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod or HIV-2 (isolate 53) wereincubated and the bound antigen was detected using a broadspectrum(anti-HIV)/horseradish peroxidase conjugate. Sera 1-7 were of Africanorigin while sera 8-11 were from Europeans. The greater ability ofAfrican sera to capture non-HIV-1 antigen can, in part, be explained bytheir higher anti-p24 titers (data not shown).

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D shows the effect of coating IgGdilution on the binding of HIV isolates.

Succesive 2-fold dilutions were made of four different sera, as shown inFIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D, beginning at a dilution of1:1000 and were used to coat microwell plates. Detergent-treatedsupernatants of HIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod and HIV-2 (isolate53) were diluted to give approximately the same optical density onplates coated with the antiserum shown in panel B at a dilution of1:1000. Bound antigen was detected using the broad-spectrum (anti-HIVIgG)/horseradish peroxidase conjugate.

FIG. 9 shows antigen capturing of virus isolates using human polyclonaland mouse anti-HIV-1 monoclonal antibodies.

Wells were coated and incubated as described in the text. The IgGs usedare as follows:

1. human polyclonal anti-HIV IgG , 2. MAb CLB 59, 3. MAb CLB 21, 4. MAbCLB 64, 5. MAb CLB 14, 6. MAb CLB 16, 7. MAb CLB 47, 8. MAb CLB 13.6(anti-p18), 9. MAb CLB 19.7, 10. MAb CLB 13.4 (anti-p18).

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, and FIG. 10E are a comparison ofthe reactivity of human anti-HIV antisera to different HIV types.

Lysates of HIV-1 (SF4), HIV-3 (ANT 70), HIV-2rod and HIV-2 (isolate 53)were separated electrophoretically on SDS-polyacrylamide gels, blottedonto nitrocellulose, and incubated with a high titer anti-HIV-1antiserum (FIG. 10A), a lower titer anti-HIV-1 antiserum (FIG. 10B),serum from the woman from whom HIV-3 (ANT 70) was isolated (FIG. 10C),her partner from which HIV-3 (ANT 70 NA) was isolated (FIG. 10D) andanti-HIV-2 antiserum from the person from whom HIV-2 (isolate 53) wasisolated (FIG. 10E).

FIG. 11A, FIG. 11B and FIG. 11C shows titrations of anti-HIV sera byenzyme immunoassay.

Microwell plates were coated with lysates of HIV-1 (SF4), HIV-3 (ANT 70)and HIV-2 (isolate 53). Serum from an HIV-1-infected European (FIG.11A), antiserum to HIV-3 (ANT 70 NA) (FIG. 11B) and antiserum to HIV-2(isolate 53) (FIG. 11C) were titrated in 2-fold dilutions beginning at adilution of 1:100 on all three coated plates.

FIG. 12A, and FIG. 12B shows the positions of methionine and tryptophanresidues in viral p17 p24 gag gene products and FIG. 12C shows thepositions of methionine and tryptophan residues in viral pol geneproducts.

Amino acid positions for the p17 gag proteins are given starting fromthe first methionine in the coding sequence. Positions for the p24 gagprotein are given starting at the p17/p24 proteolytic cleavage site.Positions for the pol gene are shown after alignment with the highlyconserved tryptophan doublet in the HIV-1 sequence at positions 556 and557. The positions of a conserved protease sequence, theprotease/reverse transcriptase cleavage site and the reversetranscriptase/endonuclease cleavage site are indicated. In this case,the terms p24 and p17 are used in the genetic sense to refer to thelargest and second largest viral core proteins respectively. The term"HIV-2 (LAV-2)" is a synonymum for HIV-2 rod.

FIG. 13A and FIG. 13B are a comparison of partial cleavage products ofgag and pol gene products of HIV-1 (SF4) [HIV-1 in the figure], HIV-3(ANT 70) [isolate 70 in the figure], HIV-2rod [HIV-2 (LAV-2) in thefigure] and HIV-2 (isolate 53) [isolate 53 in the figure]. The terms p24and p17 are used in the genetic sense to indicate the largest and secondlargest viral core proteins, respectively.

FIG. 14A-I, FIG. 14A-II, FIG. 14A-III, FIG. 14B-1, FIG. 14B-II, and FIG.14B-III shows hybridization of cDNA probes to viral RNA.

Viral RNA from HIV-1 (SF4), HIV-2rod, and HIV-3 (ANT 70) were spottedonto a membrane filter as described in Materials and Methods. Thefilters were hybridized under either nonstringent (A) or stringentconditions (B) and autoradiographed.

1. Morphology

Electron microscopy of HIV-3-infected MT4 cells revealed the presence ofextracellular virus particles having a diameter of approximately 120 nmand consisting of an outer envelope which surrounds an inner elongatedcore which has a diameter of approximately 20 to 40 nm and which appearsin some thin sections to be slightly cone-shaped in contrast to the moreor less cylindrical appearance of the HIV-1 core. Nevertheless, HIV-3 ismorphologically very similar to HIV-1 and HIV-2 but is readilydistinguished from other human retroviruses such as HTLV-I and HTLV-II.

2. Protein and Glycoprotein Antigens.

The virus present in the culture supernatant of HIV-3-infected Molt-4cells was concentrated by precipitation with polyethyleneglycol (averagemolecular weight 6000) followed by centrifugation. The resulting pelletwas resuspended in phosphate buffered saline, layered on top of a 20%sucrose cushion and pelleted at 100,000 g for 1.5 hours. The pelletedvirus was then dissociated in 62.5 mM Tris, pH 6.7, containing 2%2-mercaptoethanol, 1% sodium dodecyl sulfate and 10% glycerol and theprinciple viral antigens were separated by electrophoresis on apolyacrylamide gel (12.5%) under denaturing-conditions. Molecular weightmarkers were included on the same gel so as to provide a basis forestimating molecular weights. Once separated, the proteins wereelectrophoretically transferred to nitrocellulose paper (Western blot)which was then incubated with an antiserum derived from a personinfected with an HIV. In the initial experiments, a high titer antiserumwas used from an individual who was infected with HIV-1 and which hadbeen previously shown to crossreact with HIV-2 gag- and pol-derivedproteins. In this manner, the molecular weights of the HIV-3 gag and polgene products could be compared with those of HIV-1 and HIV-2.

The apparent molecular weights observed for the HIV-3 proteins are closeto those observed for both HIV-1 and HIV-2. Nevertheless, small yetreproducible molecular weight differences between HIV-3, and HIV-1 andHIV-2 proteins are also evident.

The protein blots revealed that HIV-3, like HIV-1 and HIV-2, possessesthree core proteins. In the case of HIV-3, these proteins were found tohave molecular weights of approximately 12,000, 16,500 and 25,000respectively. By convention, proteins are frequently referred to by a"p" for protein or "gp" for glycoprotein, followed by a number which,when multiplied by 1,000, gives the approximate molecular weight of thepolypeptide. The three major core proteins of HIV-3 will be referred tohereafter as p12, p16, and p25 respectively.

The molecular weight values as determined are expected to be correct towithin 10% of the true values. Nevertheless, much confusion exists withregards to molecular weight values of proteins since the construction ofthe electrophoresis apparatus used and the source of the buffercomponents varies from laboratory to laboratory. It is thereforenecessary when comparing the apparent molecular weights of the proteinantigens of HIV-3 with respect to those of HIV-1 or HIV-2, to subjectall samples to electrophoresis on the same gel. Such a gel can, forexample, be seen in FIG. 5. In particular, it is evident that while, inthe case of the major core protein, the molecular weight values of thehomologous proteins of the three HIVs are very close, the proteinderived from HIV-1 is the smallest. The major core protein of HIV-2 issomewhat larger then that of HIV-1, as has been previously reported. Thehomologous protein from HIV-3 is slightly larger than the major coreprotein of HIV-2. The calculated molecular weights of these proteins aregiven in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Comparison of molecular weights of gag and pol                                 gene products.                                                                                   pol     env                                                                   REVERSE TRANS  OUTER                                          TRANSCRIP- MEMBRANE MEMBRANE                                                gag ENDO TASE** PROTEIN PROTEIN                                             __________________________________________________________________________    HIV-1                                                                             12 KD 17* KD, 24 KD                                                                       31 KD                                                                             49 KD/65 KD                                                                           gp41   gp120                                        ANT 70 12 KD 16.5 KD, 24.8 KD 31 KD 48.5 KD/62 KD    gp41 gp120                                                 HIV-2 12 KD 16 KD, 24.3 KD 31 KD 53                                          KD/69 KD gp41 gp120                        __________________________________________________________________________     *Some strain to strain variation in molecular weight has been observed fo     this protein.                                                                 **Molecular weights are given for both species of reverse transcriptase. 

Similarly, molecular weight differences are also apparent between thethree HIVs with respect to the second core protein which has, in mostHIV-1 strains, a molecular weight of 18,000. Strain to straindifferences in the molecular weight of this protein have, however, beendocumented in the case of HIV-1, and the molecular weight of thisprotein may be 17,000 in some isolates. The homologous protein fromHIV-2 has a molecular weight of approximately 16,000 while the HIV-3protein has an intermediate molecular weight of approximately 16,500.

By analogy with HIV-1 and HIV-2, HIV-3 also possesses two forms of thevirally encoded enzyme reverse transcriptase. These two species alsodiffer slightly in molecular weight from the corresponding species inHIV-1 and HIV-2 and are characteristic for HIV-3. These molecularweights are also summarized in Table 1.

HIV-3 possesses an additional pol gene-derived polypeptide which is anendonuclease with apparent molecular weight of 31,000 and which does notdiffer significantly in molecular weight from the homologous proteinsfrom HIV-1 or HIV-2.

When protein blots containing HIV-3 proteins are incubated with serumobtained from an individual infected with this virus, two additionalproteins can be seen. These proteins are derived from the env gene andare the viral envelope glycoproteins. The smallest protein, which is thetransmembrane protein, migrates as a broad band with an apparentmolecular weight of between 40,000 and 45,000. This protein willhenceforth be referred to as gp41, with the understanding that theprotein exhibits some intrinsic heterogeneity with respect to itsapparent molecular weight and migration on polyacrylamide gels. Thelarger protein, which is the outer membrane protein, is similarlysomewhat diffuse on polyacrylamide gels and has a molecular weight ofapproximately 120,000. This protein will henceforth be referred to asgp120. It should be noted that the apparent heterogeneous migration ofthese two species on polyacrylamide gel is not due to hetergeneity inthe polypeptide chain but rather in posttranslational glycosylation. Inparticular, the gp120 is heavily glycosylated and the apparent molecularweight which one observes is to some degree influenced by the cell lineused to produce the virus.

In addition to the Western blot, viral protein antigens can also bevisualized by radioimmunoprecipitation assay (RIPA).

For this purpose, viral proteins can be radioactively labeledmetabolically in vivo by culturing HIV-3-infected cells in the presenceof 35S-cysteine and 35S-methionine (200 Ci/ml) in RPMI 1640 mediumdevoid of these two amino acids and supplemented with dialyzed fetalcalf serum. After 16 hours, the labeled virus is harvested from theculture supernatant by centrifugation over a 20% sucrose cushion at100,000 g for 1.5 hours. The resulting pelleted virus is thenresuspended in RIPA buffer (20 mM triethanolamine, pH 8.0, 0.5 M NaCl,0.5% Nonidet P40, 0.1% sodium deoxycholate, and 1 mMphenylmethylsulfonylfluoride).

Alternatively, the virus may be radioactivly labeled with 125 I usingchloramine T by the technique familiar to persons versed in the art. Inthis case, virus is purified from the supernatant of infected cells bypelleting the virus through a cushion of 20% sucrose, resuspending thevirus in phosphate buffered saline and banding the virus byultracentrifugation on a 20% to 50% sucrose gradient at 60,000 g for 12hours. The banded virus can be located in the fractionated gradienteither by reverse transcriptase assay or by an antigen capturing assay.The fractions containing virus are pooled and Triton X-100 is added to aconcentration of 0.5%. The Triton X-100- lysed virus may then beiodinated.

For immunoprecipitations, 100,000-200,000 cpm of labeled viral proteinin RIPA buffer is reacted with 5 microliters of a test serum in a volumeof 200 microliters for 16 hours at 4° C. The resulting immune complexesare then bound to Protein A-Sepharose (Pharmacia), washed extensively,and the bound proteins eluted with electrophoresis sample buffercontaining 1% SDS. The antigens are subsequently analyzed byelectrophoresis followed by autoradiography.

The protein antigens of HIV-3 can be characterized with respect to thoseof HIV-1 and HIV-2 using two different but related approaches. On onehand, the antigens may be characterized on the basis of their ability tocrossreact with antisera from persons infected with HIV-1 and HIV-2. Onthe other hand, antisera from persons infected with HIV-3, which containantibodies produced in response to HIV-3 antigens, can be used to testcrossreactivity to HIV-1 and HIV-2 proteins. The antigenic relationshipsbetween HIV-3, and HIV-1 and HIV-2 are substantially illustrated in theexamples given below.

The results of these experiments indicate that HIV-3 is only distantlyrelated to HIV-2 since crossreactivity is only observed with respect tothe viral core proteins and pol gene products. No crossreactivity of theenv gene products was observed when anti-HIV-2 antiserum was incubatedwith HIV-3 proteins or when anti-HIV-3 antiserum was incubated withHIV-2 proteins.

In contrast, HIV-3 is more closely related to HIV-1 since anti- HIV-3antiserum crossreacts not only with the gag and pol gene products ofHIV-1 but also to some extent with the gp41 and gp120 env gene products,albeit with a lower affinity. Anti- HIV-1 antiserum similarlycrossreacts with all of the protein antigens of HIV-3, but with a loweraffinity than for the proteins of HIV-1.

In the examples which follow, it is demonstrated that HIV-3 isantigenically substantially different from HIV-1 on the basis of 1.) adifferent pattern of reactivity with anti-HIV-1 antiserum than thatobserved for HIV-1, 2.) a drastically reduced ability to be recognizedby mouse monoclonal antibodies raised against the HIV-1 p24 and p17 coreproteins, and 3.) preferential recognition of HIV-3 proteins, includingthe envelope proteins, over HIV-1 proteins by antisera from HIV-3-infected individuals.

In spite of the genetic variation characteristic of humanimmunodeficiency viruses, a test based, for example, on HIV-1 proteinsderived from a particular strain will function satisfactorily fordetecting antibodies raised in response to other HIV-1 variants. Thiscan, in particular, also be seen in the example in which monoclonalantibodies were tested for their ability to react with antigens derivedfrom HIV-1, HIV-2 and HIV-3 isolates. In this case, the monoclonalantibodies were raised against the core proteins from the HIV-1 IIIBstrain, yet react very strongly to proteins derived from HIV-1 strainSF4. In contrast, these same monoclonal antibodies react only weakly ornot at all with HIV-3 core proteins. This indicates that the antigenicdifferences between HIV-1 and HIV-3 are of such a magnitude thatimmunological assays based on the use of HIV-1 proteins will not besuitable for testing sera from individuals infected with HIV-3.

Finally, in the examples given below, differences have been shown in thenumber and/or positions of methionine and tryptophan residues in themost highly conserved gag and pol gene products.

3. HIV-3 Nucleic Acids

A. HIV-3 Viral RNA.

The RNA of HIV-3 when deposited on a Hybond-H (Amersham) filteraccording to the "dot blot" technique, did not hybridize- to HIV-1 DNAunder stringent hybridization conditions.

By "stringent conditions" or "nonstringent conditions" are meant theconditions under which the actual hybridization and/or the subsequentwash steps are performed.

Dot blot hybridizations were performed by spotting dilutions of viralRNA from HIV-1 strain SF4, HIV-2 rod and HIV-3 strain ANT 70 ontoHybond-H filters.

The dilution series for each virus corresponded to viral RNA pelletedfrom the equivalent of 5, 2.5, 1.25 and 0.62 milliliters of culturesupernatant. The RNA was fixed onto the filter by U.V. irradiation for 2min and subjected to hybridization by bringing the filter into contactwith a 32P- labeled DNA probe. The probe chosen was derived from theHIV-1 sequence spanning nucleotides 487-4652 (Sac I-Eco R1) and includesa portion of the 5'long terminal repeat, the entire gag region and mostof the pol gene, subcloned in the vector pUC 13. Hybridization of the32P-labeled probe with the filter was carried out under stringentconditions in 3× SSC, 0.5% milk powder, 1% SDS, 10% dextran sulfate, 50%formamide (volume/volume) at 42° C. for 18 hrs (1× SSC corresponds to0.15 M NaCl, 0.015 M sodium citrate). The subsequent wash steps werecarried out under stringent conditions in 0.1× SSC and 0.1% SDS at 65°C. (2-30 minute washes). The filter was then dried and autoradiographedwith enhancing screens at -70° C. Following autoradiography, only spotswere visible which corresponded to HIV-1 viral RNA. No hybridization wasobserved to HIV-2 or either of the two HIV-3 strains. HIV-3 thereforeappears to be only distantly related to HIV-1.

B. cDNA and Subclones of cDNA Derived from HIV-3.

The conditions under which cDNA corresponding to HIV-3 sequences wassynthesized and cloned are described below. HIV-3 (strain ANT 70) from 1liter of culture was precipitated with polyethylene glycol 6000,redissolved in phosphate buffered saline, and pelleted through a 20%sucrose cushion. The resulting virus pellet was dissolved in 6 Mguanidinium chloride in 20 mM dithiothreitol and 0.5% Nonidet P-40. CsClwas added to a concentration of 2 molar and the solution containingdisrupted virus was layered onto a 1.2 milliliter cushion of 5.7 M CsClcontaining 0.1 EDTA. Viral RNA was pelleted by centrifuging for 20 hrs.at 25,000 rpm in a Beckman SW28 rotor at 15° C. The pelleted RNA wasredissolved, extracted with phenol and precipitated with ethanol and 2 MLiCl.

One-fifth of the viral RNA, prepared as described above, was used todirect the first step in the synthesis of cDNA which made use of anoligo (dT) primer which served to prime the synthesis of the first cDNAstrand.

A commercially available kit supplied by Amersham was used forpreparation of HIV-3 cDNA and made use of an exogenously added reversetranscriptase to synthesize the first strand. The synthesis of thesecond strand was performed using E. coli DNA polymerase I in thepresence of RNase H to digest away the RNA strand of the RNA/DNA hybrid.

Second strand synthesis was performed in the presence of 32p- dCTP tolabel the cDNA. The resulting cDNA was treated with T4 DNA polymerase tocreate blunt ends, the cDNA was methylated to protect possible internalEcoRI cleavage sites, and was then coupled to EcoRI linkers, alsosupplied by Amersham. The EcoRI restriction sites were then cleaved andthe cDNA was sized on a 1.2% agarose gel. The region in the gelcorresponding to a cDNA length of 500 to 2000 base pairs was excised andthe cDNA was eluted and cloned in the vector pUC13 which had beencleaved with EcoRI and dephosphorylated. The DNA was then used totransform competent cells of E. coli MC1016 (lambda). The resultingcolonies were transferred to Pall membranes (Pall Biodyne), lysed anddenatured with 1.5 M NaCl, 0.5 M NaOH and neutralized with 3 M NaOAc, pH5.5. Screening of colonies harboring an insert of HIV-3 was performedunder moderately stringent conditions in a buffer containing 5× SSC, 5×Denhardts solution, 0.2% SDS, 250 mg/ml denatured salmon sperm DNA,overnight at 65° C., using 32P-labeled plasmid containing the SacI-EcoRIfragment of HIV-1 discussed above. Following hybridization, filters werewashed as follows:

1. 1 hour in 2× SSC, 0.1% SDS at room temperature.

2. 30 minutes in 0.1× SSC, 0.1% SDS at room temperature.

3. 20 minutes in 2× SSC, 0.1 SDS at 42° C.

4. 20 minutes in 0.1× SSC, 0.1% SDS at 42° C.

Following autoradiography of the filter, several weakly positivecolonies were identified which were then grown for analysis. It wasexpected that the positive signal would either be due to weak homologywith the gag or pol regions of HIV-1, or due to some sequence homologywith the R region of the LTR.

C. Sequences Contained in HIV-3 cDNA.

A clone carrying the largest insert, which was found to be 906 basepairs in length and is referred to as iso 70-11, was selected forsequence analysis. A number of subclones of the insert were prepared bydigesting the insert with various restriction enzymes and subcloning theresulting fragments in the pUC 13 vector. Sequence determinations wereperformed according to the dideoxy-method, described by Sanger, (Proc.Natl. Acad. Sci. USA 74: 5463-5467, 1977), using a kit purchased fromBoehringer which makes use of 17-mer M13 primers. Sequence analysis ofcDNA clone iso 70-11 revealed that the insert corresponded to the 3' endof the viral genome which possessed a poly (A) chain at the 3' end.

The HIV-3 retrovirus contains a 3' LTR which is composed of a U3 regionas well as an R region. Like the 3' LTR region of HIV-1, clone iso 70-11contains an AATAAA polyadenylation signal located approximately 23nucleotides from the 3' end of the R region. Analysis of the HIV-3sequence revealed approximately 70% homology with the corresponding 3'LTR sequence of HIV-1 and less than 55% homology with the correspondingsequence of HIV-2.

Conversely, hybridizations using HIV-1 gag-pol sequences as the labeledprobe to detect crosshybridization with HIV-3 RNA revealed no detectablehybridization when the hybridization was carried out under stringentconditions. This again indicates that the viruses are only distantlyrelated and that a distinction can be made between HIV-1 and HIV-3 atthe nucleic acid level in the region of the genome encompassing the gagand pol genes. This same labeled probe did, however, hybridize to RNAderived from HIV-1 strain SF4.

In addition, the invention relates to a composition comprising at leastone antigen, in particular, a protein or glycoprotein of HIV-3retrovirus. Such a composition can be used in methods for detectingantibodies and in kits for carrying out such methods.

The HIV-3 virus has proven to be a usable as a source of antigen fordetecting antibodies in people who have come into contact with HIV-3. Assuch, the virus may be grown and concentrated by the methods alreadydescribed and a lysate prepared by treating the virus with a suitabledetergent. A preferred detergent for preparing a total viral lysate isTriton X-100, used at a concentration of 0.5%. Another preferreddetergent is Nonidet P-40 (NP-40), also used at a concentration of 0.5%.

Alternatively, viral protein may be purified from lysates of the virus.A preferred method for purifying these proteins is affinitychromatography. For example, the viral antigens may be separated on apreparative polyacrylamide gel and the individual antigens eluted inpurified form. These may further be used to raise antisera in, forexample, rabbits which are specific for the individual viral proteins.The IgG fraction derived from immune rabbit serum can be coupled to asolid phase such as CNBr-activated Sepharose 4B (Pharmacia) and used toselectively remove individual viral antigens from viral lysates. Theseproteins may then be eluted from the affinity support using a low pHbuffer and further purified using standard chromatographic techniques ofwhich an example is given by Montelaro et al., J. of Virology (1982) 42:1029-1030.

The invention relates generally to any composition which can be used forthe diagnosis of HIV-3 infection or for tests which have a prognosticvalue. These diagnostic procedures involve the detection of antibody inserum or other body fluid, which are directed against at least one ofthe antigens of HIV-3.

Preferred compositions are viral lysates or purified antigens whichcontain at least one of the viral core proteins, p12, p16, and p25 orenvelope proteins gp41 or gp120, or pol gene-derived proteins, such asp31. Especially preferred compositions are those which simultaneouslycontain, by way of example, the following proteins,

25 and gp120

p25 and gp41

p25, gp41 and gp120

p12, p16 and p25

p25, p31 and gp120

It should be understood however, that the above mentioned compositionsare only meant to serve as examples and that the invention relates toall lysates or protein preparations containing one or more of the abovementioned proteins or glycoproteins.

The invention also relates to any composition in which either HIV-3viral lysate is used in combination with similarly prepared proteinsderived from HIV-1 and/or HIV-2 for the general diagnosis of infectionor contact with human immunodeficiency virus without regard to theabsolute identity of the virus being detected. For example, suchcompositions could consist of a mixture of lysates of HIV-1, HIV-2 andHIV-3 or could consist of the following:

core proteins of HIV-1, HIV-2 and HIV-3, and in particular the majorcore protein of each virus type, homologous to the HIV-3 p25 protein.

envelope glycoproteins of HIV-1, HIV-2 and HIV-3 and in particular theouter envelope glycoproteins of each virus type, homologous to HIV-3gp120.

core proteins of HIV-1, HIV-2 and HIV-3 together with the envelopeglycoproteins of HIV-1, HIV-2 and HIV-3, in particular the major coreprotein of each virus type, homologous to the HIV-3 p25 protein,together with the major outer envelope protein of each virus, homologousto HIV-3 gp120.

a combination of the core proteins and envelope proteins of HIV-1, HIV-2and HIV-3 and in particular homologous to the HIV-3 proteins p25 andgp120 respectively and a protein derived from the pol gene of HIV-1,HIV-2 and HIV-3, in particular the proteins of each virus typehomologous to the p31 endonuclease protein of HIV-3.

Furthermore, the invention relates to an antigen providing a single bandin polyacrylamide gel electrophoresis, said antigen comprising, incommon with one of the purified antigens of HIV-3 retrovirus, an epitopethat is recognized by serum of persons carrying anti-HIV-3 antibodies.The amino acid sequences corresponding to these epitopes can readily bedetermined by isolating the individual proteins either by preparativeelectrophoresis or by affinity chromatography and determining the aminoacid sequence of either the entire protein or the fragments producedenzymatically by trypsin or chymotrypsin digestion or chemically by theprocedures described in detail below. The resulting peptide orpolypeptides can subsequently be sequenced by Edman degradation. Theinvention relates therefore to any protein, glycoprotein op peptide,either derived directly from the virus or produced by cloning any cDNAfragments of the virus in bacterial expression vectors, or viralexpression vectors for the expression of inserted DNA in mammalian orinsect cells, and purifying the expressed protein by the methodsdescribed above.

Furthermore, the invention also relates to synthetic peptides, producedeither by Merrifield synthesis or Fmoc chemistry, which may besubsequently purified to homogeneity and which contain in theirsequences epitopes which are shared by the natural HIV-3 antigens.

Antigens which share epitopes with viral proteins may easily berecognized by their reaction with antibodies present in the serum ofindividuals infected with HIV-3, either by Western blotting, orradioimmunoprecipitation. In the case of small peptides which are notable to bind to nitrocellulose, these peptides can be detected bybinding to nylon membranes (Pall Biodyne or Amersham) and reacting themembrane with anti-HIV-3 antiserum. In particular, the invention relatesto epitopes contained in any of the HIV-3 core proteins, p12, p16 andp25 or in a protein which may contain as part of its polypeptide chainepitopes derived from a combination of the core proteins. Furthermore,the invention relates to epitopes contained in either of the two HIV-3envelope glycoproteins, gp41 and gp120 as well as any protein whichcontains, as part of its polypeptide chain, epitopes derived from acombination of the HIV-3 envelope glycoprotein or a combination of theHIV-3 envelope glycoproteins and HIV-3 core protein. The inventionadditionally relates to polypeptides whose synthesis is directed byexpression vectors constructed by recombinant DNA methods whichincorporate epitopes derived from HIV-3 proteins or glycoproteinstogether with epitopes derived from the proteins or glycoproteins ofeither HIV-1 and/or HIV-2 into a single polypeptide chain. Preparingsuch a construction would involve excising the relevant coding regionsfrom cDNA of HIV-3 as well as HIV-1 and HIV-2, and coupling the DNA inphase so as to form a coding sequence which, when inserted into anexpression vector possessing the necessary signal sequences, directs thesynthesis of a hybrid protein in which epitopes of the HIV-3, HIV-1 andHIV-2 are contained.

Furthermore, the invention relates to methods for the detection ofantibodies against HIV-3 retrovirus in a biological fluid, in particularfor the diagnosis of a potential or existing ARC or AIDS caused by HIV-3retrovirus, characterized by contacting body fluid of a person to bediagnosed with a composition containing one or more of the proteins orglycoproteins of HIV-3 or with a lysate of the virus, or with an antigenpossessing epitopes common to HIV-3, and detecting the immunologicalconjugate formed between the anti-HIV-3 antibodies and the antigen(s)used.

Preferred methods include, for example, immunofluorescence assays orimmunoenzymatic assays.

Immunofluorescence assays typically involve incubating, for example,serum from the person to be tested with cells infected with HIV-3 andwhich have been fixed and permeabilized with cold acetone. Immunecomplexes formed are detected using either direct or indirect methodsand involve the use of antibodies which specifically react to humanimmunoglobulins. Detection is achieved by using antibodies to which havebeen coupled fluorescent labels, such as fluorsecein or rhodamine.

Immunoenzymatic assays may be performed, for example, as follows:

a specific quantity of HIV-3 virus extract or of a composition referredto according to the invention is deposited in the wells of amicrotitration plate.

the excess unbound material is removed after a suitable incubationperiod by washing.

a suitable dilution or dilutions of serum of other body fluid which isto be tested for the presence of antibodies directed against one or moreof the protein or glycoprotein antigen of HIV-3 is introduced into thewell.

the microtitration plate is incubated for a period of time necessary forthe binding reaction to occur.

the plate is washed thoroughly.

the presence of immune complexes is detected using antibodies whichspecifically bind to human immunoglobulins, and which have been labeledwith an enzyme, preferably but not limited to either horseradishperoxidase, alkaline phosphatase, or beta-galactosidase, which iscapable of converting a colorless or nearly colorless substrate into ahighly colored product. Alternatively, the detection system may employan enzyme which, in the presence of the proper substrate(s), emitslight.

the amount of product formed is detected either visually,spectrophotometrically, or luminometrically, and is compared to asimilarly treated control.

Other detection systems which may also be used include those based onthe use of protein A derived from Staphylococcus aureus Cowan strain I,protein G from group C Streptococcus sp. (strain 26RP66), or systemswhich employ the use of the biotin-avidin binding reaction.

Another method of immunoenzymatic detection of the presence ofantibodies directed against one or more of the HIV-3 antigens is theWestern blot. The viral antigens are separated electrophoretically andtransferred to a nitrocellulose membrane or other suitable support. Thebody fluid to be tested is then brought into contact with the membraneand the presence of the immune complexes formed is detected by themethod already described. In a variation on this methods, purified viralantigen is applied in lines or spots on a membrane and allowed to bind.The membrane is subsequently brought into contact with the body fluid tobe tested and the immune complexes formed are detected using thepreviously described techniques.

The presence of antibodies in body fluid may also be detected byagglutination. HIV-3 lysates or a HIV-3 lysate, antigen or purifiedantigen composition referred to according to this invention, is used tocoat, for example, latex particles which form an uniform suspension.When mixed with serum containing antibodies to the antigen present, thelatex particles are caused to agglutinate and the presence of largeaggregates can be detected visually.

The present invention also relates to labeled extracts of HIV-3 orcompositions as previously described. The labeling can be of any type,such as enzymatic, chemical, fluorescent or radioactive.

Furthermore, the invention relates to a method for detecting thepresence of HIV-3 antigens in body fluids. This may, for example, beaccomplished in the following manner:

the IgG fraction of antiserum, derived either from humans infected withHIV-3 or from animals injected with an HIV-3 lysate or compositionalready described, is placed in the wells of a microtitration plate.

after a suitable period to allow adsorption, the excess unbound materialis washed away.

a body fluid containing the antigen to be detected is placed in thewell.

the microtitration plate is allowed to incubate for a suitable period oftime to allow binding to occur.

the plate is then thoroughly washed with a suitable buffer.

the presence of bound antigen is detected either directly or indirectly,for example, by using immunoglobulins which are similarly specific forthe antigen(s) to be detected and which have been labeled, preferablywith one of the aforementioned enzymes.

an appropriate substrate is then added and the extent of reaction iscompared to a control in order to measure the amount of antigen present.

Furthermore, the invention relates to a kit for the detection ofanti-HIV-3 antibodies in biological fluids, comprising an HIV-3 lysateor a composition as referred to above and a means for detecting theimmunological complexes formed.

In the case of kits designed to detect specific antibodies byimmunoenzymatic methods such a kit would include:

an HIV-3 lysate or composition of one of the types already described,preferably in a purified form, and preferably attached to a solidsupport such as a microtitration plate.

a conjugate between an enzyme and an immunoglobulin fraction which iscapable of binding to the antibodies to be detected, or a conjugatebetween an enzyme and bacterial protein A or protein G.

a control antigen which possesses no epitopes which are shared by anyhuman immunodeficiency virus.

appropriate buffers for performing the assay.

an appropriate substrate for the enzyme.

Kits for the detection of specific antibodies which make use of labeledantigen would include:

an appropriately labeled antigen or combination of antigens of the typesalready described.

rotein A or anti-human immunoglobulins, preferably coupled to aninsoluble support, such as Protein A-Sepharose 4B (Pharmacia) or anequivalent support.

control antigen, which is not recognized by anti-HIV-3 antisera.

appropriate buffers for performing the assay.

if appropriate, substrates for the detection of enzymatically labeledantigen.

The invention further relates to kits, developed for the detection ofHIV-3 antigens in biological fluids, which comprise:

anti-HIV-3 immunoglobulins, preferably coupled to a solid support suchas a microtitration plate.

anti-HIV-3 immunoglobulins conjugated to an enzyme.

negative control antigen, which would not be recognized by anti-HIV-3immunoglobulins.

positive control antigen which consists of one of the HIV-3 antigens orcompositions already described.

appropriate buffers for conducting the test.

an appropriate substrate for detection of bound enzyme.

Furthermore, the invention relates to an immunogenic compositioncontaining an envelope glycoprotein of HIV-3 retrovirus, in particular,gp41 or gp 120, or a part of said glycoprotein, in combination with apharmaceutically acceptable vehicle suitable for the constitution ofvaccines effective against HIV-3. The invention additionally relates toany peptide or polypeptide which contains within its sequence all orpart of the protein backbone of the HIV-3 retrovirus, as well aspeptides which result from addition, substitution, or deletion of aminoacids which do not affect the general immunological properties of saidpeptides.

The invention further relates to monoclonal antibodies characterized bytheir ability to specifically recognize epitopes contained in the HIV-3antigens or compositions as previously defined, and in particular,monoclonal antibodies raised specifically against said antigens andproduced by traditional techniques. The invention also relates tomonoclonal antibodies of human origin produced by immortalizing B-cellsderived from persons infected with HIV-3, for example, by transformingthe B- cells with Epstein-Barr virus and subcloning the transformants.

The invention likewise relates to the production of polyclonal antiserain animals which recognize one or more HIV-3 antigens and which isproduced by infecting animals with purified HIV-3 or an HIV-3 antigen orcombination of antigens, and in particular the proteins or glycoproteinsof HIV-3.

The antibodies, either polyclonal or monoclonal, can be used for a widevariety of purposes which include neutralization of HIV-3 infectivity,the detection of HIV-3 antigens in biological fluids or in infectedcells, and the purification of HIV-3 protein and glycoprotein antigens.

The invention further relates to nucleic acids, optionally labeled whichare derived in part, at least, from RNA of HIV-3 retrovirus or ofvariants of this virus.

The invention relates likewise to the use of cDNA or parts of the cDNAor the recombinants containing them, which are characterized bycontaining at least a portion of the cDNA corresponding to the entiregenomic RNA of the HIV-3 retrovirus. Such cDNAs may be used as probesfor the specific detection of HIV-3 sequences in biological fluids,tissues and cells. The probes are preferably also labeled, eitherradioactively or chemically, alternatively using enzymatic, fluorescentor chemiluminescent labels which enable the probes to be detected.Preferred probes for the specific detection of HIV-3 and diagnosis ofHIV-3 infection are probes that contain all or a portion of the cDNAcomplementary to the HIV-3 genome. In this context, an especiallyadvantageous probe can be characterized as one which contains, inparticular, the nucleic acid sequence contained in clone iso 70-11 andwhich includes the viral LTR-R sequence which is located at both the 5'and 3' ends of viral genomic RNA and at both the 5' and 3' ends ofintegrated proviral DNA.

It is nevertheless understood that the probes which can be used for thediagnosis of HIV-3 infection are in no way limited to the probesdescribed above, and that the invention incorporates all sequences whichoriginate from the HIV-3 genome or its naturally occurring variants andincludes sequences encoding the viral core proteins (gag gene), the twoforms of reverse trancriptase and the endonuclease (pol gene), as wellas the two viral envelope glycoproteins (env gene).

The invention also relates to HIV-3 nucleic acid sequences which havebeen incorporated into a recombinant nucleic acid comprising a nucleicacid from a vector, and having said cDNA or part of said cDNA insertedtherein. Such a construction could be used for replicating the viralcDNA or its fragments in an organism or cell other than the natural hostso as to provide sufficient quantities of the probe to be used fordiagnostic purposes.

A probe generated in such a manner can be employed in a diagnostic testfor specific detection of HIV-3 which incorporates the followingessential steps:

labeling of the probe generated as described above by the methodspreviously described.

bringing the probe into contact under stringent hybridization conditionswith DNA from infected cells or viral RNA from infected cells orbiological fluids, once said DNA or RNA has been, preferably, applied toa membrane and has been rendered accessible to the probe.

washing the membrane with a buffer under circumstances in whichstringent conditions are maintained.

detection of the labeled probe, preferably by autoradiography in casesin which the probe has been radioactively labeled, or by a suitableimmunodetection technique in case the probe has been labeled chemically.

The invention further relates to a process for the production of HIV-3retrovirus characterized by culturing human T4 lymphocytes or humanlymphocytic cell lines of leukemic origin which carry the T4+ phenotypewith lymphocytes or cell lines that have previously been infected withan isolate of HIV-3 retrovirus, as well as recovering and purifying theretrovirus from the culture medium.

The invention likewise relates to a process for the production ofantigens of HIV-3 retrovirus, characterized by lysing the retrovirus,preferably with a detergent, and recovering the lysate containing saidantigens.

The invention additionally relates to a process for the production ofany of the HIV-3 proteins or glycoproteins p12, p16, p25, p31, gp41, gp120 or reverse trancriptase as previously defined, or a part thereof,characterized by inserting the nucleic acid encoding the proteins orglycoproteins in an expression vector, transforming a host with saidvector, culturing the transformed host as well as recovering andpurifying the expressed protein. The process includes vectors which mayor may not direct the synthesis of fusion proteins and includes but isnot limited to bacterial expression vectors, mammalian expressionvectors such as vaccinia virus, and vectors based on baculovirus for theexpression of cloned genes in insect cells.

EXAMPLES MATERIALS AND METHODS

Virus and Cell Culture

a. Virus Strains and Cell Lines.

HUT-78 cells chronically infected with ARV-4 (HIV-1 SF4), originallyisolated by J. Levy, San Francisco, U.S.A. (20) and uninfected HUT-78cells were kindly provided by S. Sprecher, Brussels, Belgium. LAV-2rodoriginally from L. Montagnier, Paris, and CEM cells were obtained fromJ. De Smeyter, Leuven, Belgium. Isolate 53, an HIV-2 isolate, wasisolated in this laboratory (21).

b. Virus Isolations.

Virus isolations were performed in a manner similar to that described byLevy and Shimabukuro (22), with modifications. Lymphocytes from patientsas well as from healthy donors were isolated from heparinized wholeblood on Lymphoprep (Nyegaard and Co., Oslo, Norway) and were culturedin RPMI 1640 containing 20 mM HEPES, 15 percent fetal calf serum(Gibco), 5 g/ml hydrocortisone (Merck), 75 U/ml IL-2 and 2 g/mlpolybrene (Aldrich).

Lymphocytes from healthy donors were stimulated with 2 g/mlphytohemagglutinin (PHA, Wellcome) for 3 days prior to use. FreshPHA-stimulated lymphocytes were added to the virus isolation culturesevery 3 to 4 days. Cultures were monitored for cytopathic effect,immunofluorescence, using a broad specificity, polyclonal referenceantiserum (23), and the presence of antigen in the culture supernatants(Innotest VCA- HIV, Innogenetics). The broad specificity reference (BSR)anti-serum used was derived from an HIV-1-infected donor and was shownexperimentally to have an exceptionally high titer (≧1,000,000 in anenzyme immunoassay based on recombinant HIV-1 p24 protein) and tocrossreact strongly with the gag and pol gene products of other HIVtypes, in particular, HIV-2. Reverse transcriptase was also assayedessentially as described (24).

In order to establish chronically infected, permanent cell lines,virus-infected primary lymphocytes were co-cultured with Molt 4 clone 8cells (25), kindly provided by N. Yamamoto, Yamaguchi, Japan, andmonitored for cell growth. Virus production was monitored by the reversetrancriptase assay as well as antigen capturing.

Differential Antigen Capturing.

A test system was developed whereby a distinction can be made betweenHIV-1 and other related human immunodeficiency viruses. The system isbased on a comparison of the ability of two different polyclonal IgGpreparations, one with a broad anti-HIV specificity which is due itsexceptionally high titer, particularly against the major core protein,and one with a lower titer which reacts preferentially with HIV-1, tocapture detergent-treated virus in culture supernatants. Detection ofcaptured antigen is achieved by using a (broad specificityIgG)/horseradish peroxidase conjugate.

The test detects primarily but not exclusively the p24 core protein.

Monoclonal Antibodies to HIV-1

The panel of monoclonal antibodies used has been described (26). Theantibodies were prepared against native viral proteins in TritonX-100-disrupted HIV-1 preparations.

Protein Analysis

a. Electrophoresis.

Polyacrylamide gel electrophoresis of viral proteins was performedessentially as described by Maizel (27).

b. Protein Blotting.

Blotting was performed either in a Bio-Rad transblot cell at 400 mA for4 hours using the carbonate buffer described by Dunn (28) or using theLKB semi-dry blotting apparatus at 0.8 mA/cm2 for 1 hour in 48 mM Tris,39 mM glycine, 0.0375% sodium dodecylsulfate (SDS) and 20% methanol.

c. Generation of Partial Cleavage Products.

Viral proteins were analyzed by the technique shown in FIG. 1. Advantagewas taken of the fact that corresponding proteins from the variousisolates have similar molecular weights. Proteins were separated on 12.5percent SDS- polyacrylamide gels together with a marker lane of ARV-4proteins which was excised following electrophoresis, blotted andincubated with an anti-HIV antiserum to reveal the positions of theviral proteins.

The marker blot was in turn used to locate the approximate positions inthe Coomassie blue stained portion of the gel of the viral proteins tobe cleaved. Horizontal gel slices containing the proteins were excised,transferred to glass tubes and subjected to chemical cleavage.

1. Cyanogen Bromide Cleavage

The gel slice was incubated with 10 ml of a freshly prepared 40 mg/mlsolution of CNBr (Merck) in 0.3 N HCl for 3 hours at room temperature ina fume hood. Following the incubation, the gel slice was equilibratedwith SDS-sample buffer for electrophoresis in the second dimension.

2. BNPS-Skatole Cleavage.

The gel slice was incubated with 10 ml of a freshly prepared saturatedsolution of 2-(-2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolinine(BNPS-Skatole, Pierce) in 70 percent acetic acid; 30% H₂ O containing0.1% phenol, for 3 hours at room temperature, protected from light.Following the incubation, the gel slice was equilibrated by repeatedwashing in SDS-electrophoresis sample buffer.

Following cleavage, the individual lanes were excised from the gelslices, rotated 90° and placed on top of a 10 to 20 percentSDS-polyacrylamide gradient gel. On completion of electrophoresis, thegel was blotted onto nitrocellulose (Schleicher and Schuell) and blockedwith PBS containing 1 mg/ml casein (Merck). Only cleavage products withmolecular weights in excess of 10 kD are able to be visualized sincepeptides with lower molecular weights do not bind efficiently tonitrocellulose. Blots were incubated with a broad spectrum anti-HIVantiserum followed by goat anti-human IgG: alkaline phosphataseconjugate (Promega).

Partial cleavage products were then visualized by reaction with5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium (Sigma).

Viral Nucleic Acids

a. Hybridization to Viral RNA.

Virus from culture supernatants was harvested by pelleting throughcushions of 20% sucrose by centrifucation at 26,500 rpm for 1.5 hrs. at4° C. and was disrupted in 10 mM Tris, pH 7.4, 10 mM NaCl, 10 mM EDTAcontaining 0.5% sodium dodecylsulfate. Aliquots of the disrupted viruswere spotted onto a membrane of Hybond H (Amersham) in amountscorresponding to 5, 2.5, 1.25 and 0.62 milliliters of original culturesupernatants. The RNA deposited onto the filter was fixed to themembrane by irradiation with ultraviolet light for 2 hrs. The RNA boundto the filter was then subjected to hybridization with an HIV-1 cDNAprobe which had been labeled by nick translation with 32p-dCTP. Thehybridization was carried out under stringent conditions in 3× SSC, 0.5%milk powder, 1% SDS, 10% dextran sulfate and 50% formamide at 42° C. for18 hrs. Following hybridization, the filter was washed twice understringent conditions in 0.1× SSC and 0.1% SDS for 30 minutes.Hybridization was detected by autoradiography at -70° C. with enhancingscreens. Hybridizations were similarly performed using a probe derivedfrom the env region of HIV-2.

Hybridizations were also performed under nonstringent conditions in 5×SSC, 25% formamide, 5× Denhardts solution, 10% dextran sulfate, and 100g/ml denatured salmon sperm DNA at 37° C. overnight. The filter wassubsequently washed 4 times for 15 minutes in 5× SSC, 0.1% SDS at roomtemperature and autoradiographed.

b. Preparation of ANT 70 cDNA

Virus was pelleted from 1 liter of culture supernatant usingpolyethylene glycol 6000, redissolved in PBS and pelleted through a 20%sucrose cushion. The resulting pellet of virus was disrupted in 6 Mguanidinium chloride in 20 mM sodium phosphate buffer, pH 6.5,containing 20 mM dithiotreitol and 0.5% NP-40. Solid CsCl was added to aconcentration of 2 molar. The solution containing disrupted virus waslayered onto a cushion of 5.7 M CsCl containing 0.1 M EDTA and the viralRNA was pelleted by centrifugation at 25,000 in a Beckman SW 28 rotor at15° C. for 20 hrs.

Following centrifugation, the RNA was redissolved, extracted with phenoland precipitated with ethanol and 2 M LiCl.

One-fifth of the viral RNA prepared was used to direct the first step inthe synthesis of cDNA using a kit supplied by Amersham. cDNA synthesiswas primed using oligo (dT). The synthesis was carried out using thereverse trancriptase supplied with the kit. Second strand synthesis wasperformed using E. coli DNA polymerase I in the presence of RNase H todigest away the RNA strand of the RNA/DNA hybrid. The synthesis of thesecond strand was performed in the presence of 32P-dCTP to label thecDNA. The resulting cDNA was treated with T4 DNA polylmerase to createblunt ends, the cDNA was methylated to protect possible internal EcoRIcleavage sites, and was then coupled to EcoRI linkers (Amersham). TheEcoRI sites in the linkers were then cleaved and the cDNA was sized on a1.2% agarose gel. The region of the gel corresponding to a cDNA lengthof 500 to 2000 base pairs was excised, and the cDNA was eluted andcloned in the vector pUC13 which had previously been cleaved with EcoRIand dephosphorylated. After ligation, the DNA was used to transformcompetent cells of E. coli MC1016 (lambda). The resulting colonies weretransferred to Pall membrane filters (Pall Biodyne), lysed and denaturedwith 1.5 M NaCl, 0.5 M NAOH and neutralized with 3 M NaOAc, pH 5.5.Screening of colonies harboring an insert of HIV-3 was carried out byhybridization under moderately stringent conditions in 5× SSC, 5×Denhardts solution, 0.2% SDS, 250 mg/ml denatured salmon sperm DNAovernight at 65° C. Hybridization was performed using the HIV-1SacI-EcoRI fragment. Following hybridization, the filters were washed asfollows:

1. 1 h. in 2× SSC, 0.1% SDS at room temperature.

2. 30 minutes in 0.1× SSC, 0.1% SDS at room temperature.

3. 20 minutes in 2× SSC, 0.1% SDS at 42° C.

4. 20 minutes in 0.1× SSC, 0.1% SDS at 42° C.

After washing, the filters were autoradiographed at -70° C. usingintensifying screens.

Hybridizations were also performed under the nonstringent conditionsused for nonstringent hybridization of the HIV-1 and HIV-2 probe.

c. Analysis of cDNA Clones.

Colonies giving a positive hybridization signal were grown for analysis.Plasmids were isolated, cleaved with EcoRI and subjected to agarose gelelectrophoresis to confirm the presence of an insert and to determineits size. Of 96 colonies analyzed 17 were found to contain inserts. Fivewere taken for further analysis and ranged in size from approximately800 to 1600 base pairs in length.

d. Sequence Determinations.

Nucleotide sequence determinations were performed according to thedideoxynucleotide method of Sanger (Proc. Natl. Acad. Sci. USA 74:5463-6467, 1977), using a kit supplied by Boehringer. Sequencing wascarried out using 17-mer M13 primers.

e. Hybridizations of ANT 70 cDNA to HIV-1 and HIV-2 Viral RNA.

The ANT 70 cDNA clone containing the largest insert (iso 70-11) was usedfor hybridization to the filter onto which viral RNAs had beendeposited.

Hybridization was performed under stringent conditions in 3× SSC, 0.5%milk powder, 1% SDS, 10% dextran sulfate, and 50% formamide at 42° C.for 18 hrs. Following hybridization, the filter was washed with 0.1×SSC, 0.1% SDS at 65° C. (2-30 minute washes) after which the filter wasautoradiographed at -70° C. with an intensifying screen.

Results

Virus Isolation

As part of a continuing study on heterosexual transmission of HIV, avirus isolation was performed from blood from a Camerounian woman andher partner. As before, the two isolated strains will be named HIV-3(ANT 70) (woman) and HIV-3 (ANT 70 NA) (man), respectively. Forconvenience, the shorter terms ANT 70 and ANT 70 NA will also be used.The woman is the partner of an HIV- seropositive man with generalizedlymphadenopathy. Serum from the woman was moderately positive (ratioO.D./cut-off of 4.5) in the enzyme-linked immunosorbent assay (EIA,Organon Teknika) and had a low titer (1/40) in the immunofluorescentantibody assay for HIV-1 but gave ambiguous results in the HIV-1 Westernblot assay with clear bands at p33, P53/55 and p64 but very weak bandsat p24, gp41 and gp120. The woman had elevated serum IgG and IgM levelsand a CD4/CD8 ratio of 0.46. Virus was isolated by co- cultivation ofthe woman's lymphocytes with PHA-stimulated lymphocytes from healthyuninfected donors. After 52 days in culture, virus was detected in theculture as judged by the presence of syncytia and on the basis ofpositive immunofluorescence observed when a laboratory referenceanti-HIV antiserum was incubated with acetone-fixed cells from theculture. The presence of reverse transcriptase was also detected in theculture supernatant (10 4 cpm/ml, 27× background). Cell-free culturesupernatant was used to passage the virus on fresh lymphocytes. After 15days, CPE was again observed and reverse transcriptase detected in thesupernatant. A comparison of detergent-treated culture supernatant fromthis isolate (ANT 70) with other isolates by differential antigencapturing revealed, however, that this isolate was not HIV-1.

These results are illustrated in FIG. 2. It is evident by the lower O.D.values that the isolate (ANT 70) is, in contrast to the other isolates,poorly recognized by the HIV-1 specific IgG but, like the otherisolates, was readily captured by the broad specificity IgG (panel F).The other isolates, which were subsequently all shown to be HIV-1strains using an HIV-1 specific MAb (CLB MAb 14), all gave higher O.D.values on the plates coated with specific IgG than on plates coated withthe broad specificity reference IgG.

An attempt was made to transfer the virus to a permanent cell line byco-cultivating isolate (ANT 70)-infected primary lymphocytes with Molt-4clone 8 cells. In the initial phase of the infection, extensivecytopathic effect was observed with syncytium formation and cell death.

Within several weeks, cell growth was detected. The cells gave apositive immunofluorescence when tested using a broad spectrum anti-HIVantiserum and the presence of antigen and reverse transcriptase waseasily detectable in the culture supernatant.

Virus was similarly isolated from the partner of the woman from whomisolate (ANT 70) was isolated (strain ANT 70 NA). The man was sufferingfrom lymphadenopathy and was classified as class 3 according to the CDCclassification system. The man also had elevated serum IgM and IgGlevels and a CD4/CD8 ratio of 0.4. Virus was detected in the supernatantof the culture on day 18. Detergent-treated supernatant containing thisvirus was also analyzed by differential antigen capturing and found toreact in a manner similar to isolate (ANT 70) (FIG. 3). The binding ofantigen derived from this isolate was again less with HIV-1 specific IgGthan with the broad specificity IgG.

Serum from the person from whom the isolate (ANT 70 NA) was derived wasincubated with HIV-1 and HIV-2 Western blot strips (Biotech). Additionalstrips were also incubated with serum from a donor infected with HIV-1as well as serum from the person from whom HIV-2 (isolate 53) wasisolated. These results are shown in FIG. 4. Serum from the personinfected with ANT 70 NA crossreacted to a significant extent withvirtually all HIV-1 proteins, including the envelope proteins. Incontrast, serum from the HIV-2-infected individual crossreacted onlywith the gag p24 protein, p34 endonuclease and p68 reversetranscriptase. The anti-HIV-1 serum recognized only the p26 gag proteinof HIV-2, while serum from the carrier of ANT 70 NA recognizes thisprotein and the HIV-2 reverse transcriptase.

Characterization of Viral Proteins.

Virus in the culture supernatant was precipitated using polyethyleneglycol 6000 (Merck) and the resulting material was redissolved andpelleted through a 15 percent sucrose cushion. The pelleted virus wasdissociated in SDS-sample buffer and analyzed by polyacrylamide gelelectrophoresis followed by protein blotting. The blot, shown in FIG. 5,was incubated with a broad specificity anti-HIV serum to reveal theviral proteins.

In addition to reacting with all of the HIV-1 viral proteins, the BSRantiserum also crossreacts with the gag and pol gene products of HIV-2.This antiserum clearly recognizes the gag and pol gene products of ANT70 as well. It is evident that the molecular weights of the ANT 70 geneproducts differ from those of either HIV-1 or HIV-2. The molecularweights of the various viral proteins are summarized in table 1. Thevariability in the HIV-1 p17/p18 protein is due to a 6 amino acidinsertion which is present in some strains between positions 120 and 121in the HIV-1 HXB2 sequence. A comparison of the proteins from ANT 70 andANT 70 NA are shown in FIG. 6. The molecular weights of all of theproteins of ANT 70 NA are identical to those of ANT 70.

In order to investigate further the antigenic relationship betweenHIV-1, HIV-2 and ANT 70, a series of African and European anti- HIV-1sera were diluted 1:1000 and used to coat microwell plates for antigencapturing.

Detergent-treated culture supernatant containing HIV-1, ANT 70, HIV-2(LAV-2rod) and HIV-2 (isolate 53) were diluted and the ability of eachantiserum to capture the four different isolates was analyzed.Representative results are shown in FIG. 7. It can be seen from thisexperiment that the ability of the various sera to capture HIV-1 is inno way related to their ability to capture either HIV-2 or ANT 70. Incontrast, the ability of these sera to capture LAV-2rod, the prototypeHIV-2 strain, is strongly correlated with the ability of these sera tocapture isolate 53, which is also an HIV-2 strain but an independentisolate. These data indicate that ANT 70 is neither HIV-1 nor HIV-2.

In a series of related antigen capturing experiments, four Africananti-HIV-1 sera were chosen in order to access their ability to bindHIV-1, ANT 70, HIV-2 (LAV-2rod) and HIV-2 (isolate 53) when the IgGswere coated at different dilutions. Culture supernatants were diluted soas to give approximately the same optical density when captured onplates coated with the IgG used in panel B of FIG. 8. Dilutions of thefour sera were coated and virus-containing supernatant was added. Theassumption was made that similar viruses should give rise to similartitration curves. Indeed, in FIG. 5, LAV-2rod and isolate 53 both reactsimilarly with the coated IgGs. On the other hand, ANT 70 gave moreintense signals at higher IgG dilutions than did either of the HIV-2isolates and the shapes of the curves obtained with ANT 70 resemble moreclosely the curves obtained for HIV-1, except that the optical densitiesare consistently lower.

Cross Reactivity of Mouse Monoclonal Antibodies Directed Against HIV-1p24 Core Protein.

A panel of mouse monoclonal antibodies (MAbs) prepared against the HIV-1p24 core protein was tested for their ability to crossreact with ANT 70and HIV-2 isolates. In principle, any panel of anti- HIV-1 p24monoclonal antibodies can be used, as long as the series includesmonoclonal antibodies which react with different epitopes on the HIV-1p24 molecule. Ascites fluid containing the antibodies was diluted andused to coat microwell plates. Detergent-treated, virus-containingsupernatants were then added to the coated wells. Bound antigen wasdetected using BSR-HIV IgGs conjugated to horseradish peroxidase. Theresults obtained are shown in FIG. 9.

In control wells coated with polyclonal broad spectrum IgGs, allvirus-containing supernatants gave optical densities which exceeded thelimits of the microwell plate reader. However, when tested in wellscoated with the various monoclonal antibodies, quite a different patternemerged. Previous studies indicated that all of the MAbs tested reactagainst different epitopes on the p24 molecule with the exception ofMAbs CLB 59 and CLB 21 which have been shown to recognize the sameepitope. Both of these two MAbs react strongly with HIV-1 as expectedand also give a measurable signal with ANT 70 but fail to react witheither of the HIV-2 strains. Two other MAbs, CLB 64 and CLB 14, boundHIV-1 well and showed a weak affinity for ANT 70 as well as the twoHIV-2 isolates. In particular, MAb CLB 14 has been shown to recognizeall HIV-1 isolates well (>150 tested). This MAb must therefore bind to avery highly conserved epitope, remnants of which can also be detected inother human immunodeficiency viruses. The other MAbs to p24 (CLB 16, 47and 19.7) and two others which were raised against the HIV-1 p18 protein(CLB 13.4 and CLB 13.6), failed to recognize either ANT 70 or the twoHIV-2 isolates but did capture the corresponding HIV-1 antigens.

Reaction of Human Anti-HIV Antisera to Viral Proteins.

Protein blots of viral proteins from HIV-1 (ARV 4), ANT 70 and HIV-2(LAV2rod) were prepared after electrophoresis of detergent-solubilizedextracts and incubated with various human sera (FIG. 10). Panel A showsthe reaction of the broad specificity laboratory reference serum withthe three virus isolates. In panel B, an anti-HIV-1 antiserum wasincubated with the blot and recognizes preferentially HIV-1 proteins.Serum from the woman from whom ANT 70 was isolated (panel C) and herpartner from whom ANT 70 NA was isolated (panel D) were tested for theirability to recognize other viral isolates. Both of these serapreferentially recognize ANT 70 including the gp120 envelope protein ofthis virus. Serum from the partner has a higher titer than serum fromthe woman from whom ANT 70 was isolated and recognizes the gp41 ofHIV-1. Both of these sera have a higher affinity for ANT 70 than forHIV-1 or the HIV-2 isolates. In contrast, serum from the person fromwhom HIV-2 isolate 53 was isolated binds preferentially to HIV-2proteins and recognizes the HIV-2 gp120 envelope protein of this virusas well as the gp41 transmembrane protein (panel E). It does not reactwith glycoproteins of HIV-1 or ANT 70. These results further indicatethat ANT 70 is different from either HIV-1 or HIV-2.

Enzyme immunosorbent assays using coated viral proteins titrations ofanti-HIV-1, anti-ANT 70 and anti-HIV-2 sera were performed in microwellplates coated with HIV-1 (ARV-4), ANT 70 and HIV-2 (isolate 53) virallysates. Two-fold dilutions of each sera, beginning at an initialdilution of 1:100, were tested for their ability to bind to the coatedantigen. Bound antibody was detected using a horseradishperoxidase-labeled goat anti-human IgG conjugate. These results areshown in FIG. 11. The anti-HIV-1 serum recognized preferentially theHIV-1 proteins but shows a significant amount of crossreaction with ANT70 proteins. The HIV-2 proteins were barely detected. In contrast,anti-ANT 70 serum preferentially recognized ANT 70 proteins, showedcrossreactivity toward HIV-1 proteins, and reacted better with the HIV-2coated wells than did the anti-HIV-1 serum as evidenced by the higheroptical density values obtained. The anti-HIV-2 serum had a very lowtiter but nevertheless reacted best with HIV-2 proteins. No detectablesignal was observed on HIV-1 or ANT 70 coated wells. The inability todetect crossreaction in this instance is undoubtedly related to the lowanti-HIV titer of this serum.

Analysis of Partial Chemical Cleavage Products of Viral Proteins.

The two reagents used for chemical cleavage, cyanogen bromide andBNPS-skatole, were chosen because of their high specificities formethionone (29) and tryptophan (30), respectively. These two amino acidsare also rather hydrophobic and are therefore also less likely to foundlocated in epitopes on the outer surfaces of protein molecules (31).Examination of published amino sequences of the gag and pol geneproducts of HIV-1 (32-36), HIV-2 (19), SIVagm (10), SIVmac (9), equineinfectious anemia virus (EIAV,37) and Visna (38) reveals that whilethere is little amino acid homology between some of these diverseisolates, many of the positions of the methionine residues in theseproteins and, to an even greater extent, the tryptophan residues, arestrikingly conserved (FIG. 12). Futhermore, intraspecies variation inthese residues is minimal or absent, at least in the case of HIV-1 (36)and probably holds true for all of the human and simian immunodeficiencyretroviruses.

The partial digestion patterns of the gag and pol gene products ofHIV-1, ANT 70, HIV-2 (LAVrod) and HIV-2 (isolate 53) are shown in FIG.13.

Inspection of the CNBr cleavage patterns of the p24 protein from thefour isolates reveals that the patterns generated for HIV-2 (LAV-2rod)and HIV-2 (isolate 53) are identical. Different patterns, however, areobserved for HIV-1 and for ANT 70. Thus, significant differences existin the locations of the methionine residues in the major core protein ofHIV-1, ANT 70 and HIV-2. In the case of the p17 core protein,differences are observed between the two HIV-2 isolates. Inspection ofthe published sequence for HIV-2rod indicates that there is a methioninelocated 18 amino acids from the carboxyl terminus of this protein. Weconclude that this methionine must be absent in the correspondingprotein from isolate 53. From the cleavage pattern it is also possibleto deduce the presence of a methionine near (10-15 amino acids) one ofthe termini of the p16 from ANT 70. CNBr cleavage of the retroviralreverse transcriptase reveals that again, the proteins from the twoHIV-2 isolates are identical, while different patterns are observed forboth HIV-1 and ANT 70 proteins. In the case of the p31 endonucleasederived from the 3'-portion of the pol gene, similarities can be deducedbetween all of the isolates although some minor differences areapparent.

BNPS-skatole cleavage of the p24 proteins from the four isolates resultsin strikingly similar patterns. It is evident from FIG. 8 that this isto be expected since the tryptophan positions in this protein are veryhighly conserved, particularly for the retroviruses of human and simianorigin. We conclude that the tryptophan positions in the ANT 70 p25protein also conform to this pattern. Inspection of the patternsreveals, however, that minor differences can be observed, not in theoverall appearance of the pattern but rather in the apparent molecularweights of the species generalized by cleavage. In particular,differences are detected in the apparent molecular weights of thecentral spots in each pattern. As expected, the patterns for HIV-2(LAV-2rod) and HIV-2 (isolate 53) are identical. The central spot in thepattern for ANT 70 has however, a larger apparent molecular weight whilethe central spot for HIV-1 (ARV-4) has a lower molecular weight. Inregard to the p16, the positions of the tryptophans in the ANT 70protein appear to resemble more closely the positions of the tryptophansfound in the HIV-2 protein. The HIV-1 p17 has a tryptophan located 16amino acids from the amino terminus of the protein and gives rise to anadditional spot not seen in the ANT 70 and HIV-2 patterns followingBNPS-skatole cleavage. The tryptophan corresponding to the one atposition 36 in the HIV-1 p17 sequence is conserved in all isolates.

The patterns generated by cleavage of the reverse transcriptase from thefour isolates are complex but is is once again apparent that the twoHIV-2 isolates are identical. Patterns are obtained for ANT 70 whichcorresponds neither to the pattern obtained for HIV-1 nor to the HIV-2pattern. Differences in apparent molecular weights of the cleavageproducts of the p31 endonuclease are also observed but the patternsgenerated from the corresponding proteins from HIV-1, ANT 70, and HIV-2also show common features which suggests a conserved structure.

Results

Viral Nucleic Acids

a. Hybridization of HIV-1 and HIV-2 cDNA to Viral RNAs.

Nucleicacids crosshybridization between HIV-1 and RNA from the virusesHIV-2 and ANT 70 was evaluated by performing the hybridization with theSacI-BglII HIV-1 restriction fragment which had been inserted into thevector pUC13. This fragment contains a portion of the 5' LTR, includingthe R region, the entire gag gene and most of the pol gene of HIV-1.Under stringent hybridization conditions, hybridization was onlyobserved between this probe and the RNA derived from HIV-1 (SF4). Nohybridization was observed between the probe and either HIV-2 or ANT 70(FIG. 14). This indicates that the gag and pol regions of HIV-2 and ANT70 are significantly different from the corresponding region of HIV-1.

The HIV-2 probe used contains a sequence of approximately 1000 basepairs derived from the env gene of HIV-2. This probe hybridized only toHIV-2 RNA under stringent hybridization conditions and no hybridizationwas observed with either HIV-1 or HIV-3.

b. Homology Between ANT 70 cDNA and Sequences of HIV-1 and HIV-2.

The cDNA cloneiso 70-11 was used as a probe to assess the degree ofnucleic acid homology between the various virus isolates. The filteronto which aliquots of HIV-1, HIV-2 and ANT 70 had been deposited wassubjected to hybridization under stringent conditions. The results arealso shown in FIG. 14. The experiment demonstrates that under stringenthybridization conditions, no crosshybridization can be detected betweenany of the virus isolates. The ANT 70 derived probe hybridizes only toANT 70.

c. Sequence Analysis of Clone iso 70-11.

Subclones of the insert were made in pUC13 and sequenced using thedideoxynucleotide method:

The presence of a poly A tail confirmed that the iso 70-11 insert isderived from the 3' end of the viral RNA.

Adjacent to the poly A tail is the sequence corresponding to the Rregion of the viral 3' LTR. Sequence contained in the ANT 70 cDNA andthe viral sequences to which they correspond are shown below:

1. HIV-3 LTR

            10         20         30         40         50         60                                                      CCCATGGATT                                                                    TGAAGATACA CATAAAGAAA TACTGATGTG                                             GAAGTTTGAT AGATCTCTAG                    -         70         80         90        100        110        120                                                 GCAACACCCA TGTTGCTATG ATAACTCACC                                             CAGAGCTCTT CCAGAAGGAC TAAAAACTGC                                                -        130        140                                                     150        160        170                                                     180                                     TGACCTGAAG ATTGCTGACA CTGTGGAACT TTCCAGCAAA GACTGCTGAC ACTGCGGGGA                                                     -        190        200                                                     210        220        230                                                     240                                     CTTTCCAGTG GGAGGGACAG GGGGCGGTTC GGGGAGTGGC TAACCCTCAG AAGCTGCATA                                                     -        250        260                                                     270        280        290                                                     300                                     TAAGCAGCCG CTTTCTGCTT GTACCGGGTC TCGGTTAGAG GACCAGGTCT GAGCCCGGGA                                                   |                                                 U3    ←→   R                                    -        310        320        330        340        350        360                                                 GCTCCCTGGC CTCTAGCTGA ACCCGCTCGT                                             TAACGCTCAA TAAAGCTTGC CTTGAGTGAG                                                Polyadenylation signal                A - POLY A                                                                    ←|                                                        

2. Sequences from HIV-3 3'ORF

    __________________________________________________________________________             10       20       30         40        50        60                    AACATGGGAAACGCATTGAGAAAAGGTAAATTTGAGGGATGGGCAGCAGTAAGAGAAAGA                  AsnMetGlyAsnAlaLeuArgLysGlyLysPheGluGlyTrpAlaAlaValArgGluArg                   -          70       80       90        100       110       120              ATGAGAAGAACTAGAACTTTCCCTGAGTCTGAACCATGCGCACCTGGAGTAGGACAGATC                  MetArgArgThrArgThrPheProGluSerGluProCysAlaProGlyValGlyGlnIle                   -         130      140      150        160       170       180               TCCAGGGAATTAGCAGCTAGAGGAGGGATACCAAGTTCCCATACTCCTCAAAACAATGCA                  SerArgGluLeuAlaAlaArgGlyGlyIleProSerSerHisThrProGlnAsnAsnAla                   -         190      200      210        220       230       240               GCCCTTGCATTCCTAGAAAGTCACCAAGAGGAAGAAGTAGGTTTTCCAGTAGCACCTCAA                  AlaLeuAlaPheLeuGluSerHisGlnGluGluGluValGlyPheProValAlaProGln                   -         250      260      270        280       290       300               GTGCCTCTAAGGCCAATGACCTATAAAGGAGCATTTGACCTCAGCTTCTTTTTAAAAGAA                  ValProLeuArgProMetThrTyrLysGlyAlaPheAspLeuSerPhePheLeuLysGlu                   -         310      320      330        340       350       360               AAGGGAGGACTGGAAGGGTTAATTTACTCCCATAAAAGAGCAGAAATCCTGGATCTTTGG                  LysGlyGlyLeuGluGlyLeuIleTyrSerHisLysArgAlaGluIleLeuAspLeuTrp                   - GTGTATAA                                                                   ValTyr                                                                       __________________________________________________________________________

Discussion

We have isolated a novel human immunodeficiency-associated retrovirusfrom a Camerounian woman (ANT 70) and her partner (ANT 70 NA). At thetime the original virus isolation was performed, the woman was onlyslightly seropositive, gave ambiguous results in the western blot testand was clinically asymptomatic. Since that time, the woman has begun todevelop some of the symptoms of AIDS-related complex (ARC). In contrast,her partner, from whom we were also able to isolate a virus with thesame characteristics as the original isolate, was suffering fromlymphadenopathy and has since developed other symptoms characteristic ofAIDS. This novel isolate may therefore be considered to be a humanimmunodeficiency virus. The fact that this same virus could be isolatedfrom sexual partners also suggests a mode of transmission which issimilar to that of human retroviruses.

The virus was first recognized as being different from HIV-1 on thebasis of its altered ability to be captured in a differential antigencapturing assay. This has proven to be a highly reliable test which isable to distinguish between HIV-1 and non-HIV-1 strains. That thisisolate is not HIV-1 is borne out at the protein level by 1.) thediffering molecular weights of the viral proteins, 2.) a differentpattern of crossreactivity with anti- HIV-1 antiserum than HIV-1, 3.) adrastically reduced ability to be recognized by mouse monoclonalantibodies raised against HIV-1 p24 and p17 core proteins, 4.)preferential recognition of ANT 70 proteins over HIV-1 proteins byantisera from the virus carrier, and 5.) patterns of partial cleavage offour of the most highly conserved viral proteins which do not match thepatterns obtained when HIV-1 proteins are subjected to the sametreatment. Nevertheless, sera from the two individuals infected withthis virus recognize the HIV-1 gp41 envelope protein. By the samecriteria listed above, it is also clear that ANT 70 is not HIV-2.Indeed, the antigenic differences between ANT 70 and HIV-1 are smallerthan those between HIV-2 and HIV-1. This is particularly evident fromthe results presented in FIGS. 8 and 10.

Additional compelling evidence that ANT 70 is a unique virus differentfrom HIV-1 and HIV-2 comes from the partial peptide maps. We have shownthat there are significant differences in the most highly conservedviral proteins. The two HIV-2 isolates which were used for comparisongave essentially identical cleavage patterns except in the case of CNBrcleavage of the p17 core protein. It should be noted, however, that thep17 core protein exhibits more variability than the p24 protein, atleast in HIV-1 strains (34). Whether or not this also holds true forHIV-2 awaits sequence determination on more strains than have beenanalyzed to date.

In light of the fact that ANT 70 is antigenically more closely relatedto HIV-1 than is HIV-2, as evidenced by a higher degree ofcrossreactivity which extends even to the gp41 envelope protein, it wasessential to establish that ANT 70 was more than simply a geneticvariant of HIV-1. This was possible by investigating the locations ofsome of the most highly conserved amino acids in a number of viralproteins which are least subject to genetic variation. That majordifferences were noted in the cleavage patterns indicates that HIV-1,HIV-2 and ANT 70 are three genetically distinct viruses. On the otherhand, the same series of experiments also revealed similarities betweenthe viruses which may indicate that all three arose from a commonprogenitor.

The hybridization data also support the notion that ANT 70 isfundamentally different from either HIV-1 and HIV-2. As long as theconditions under which the hybridization is performed are stringent, adistinction can easily be made between the three virus types. RNA of theHIV-3 retrovirus virtually hybridizes neither with the Env gene or theLTR close to it, in particular not with the nucleotide sequence8352-9538 of HIV-1, nor with the sequences of the Pol region of theHIV-1 genome under stringent conditions.

Analysis of the cDNA sequences revealed that the insert is derived fromthe 3' end of the viral genome. An analysis of the homology betweenthese sequences and the sequence of HIV-1 and HIV-2 reveal that ANT 70is somewhat more closely related to HIV-1, particularly in the LTRsequences (approx. 70% homology). The differences are nevertheless ofsuch magnitude as to rule out the possibility that ANT 70 is simply agenetic variant of HIV-1. The ANT 70 3' LTR also contains the signalsequences which are typical of retroviral LTRs.

The existence of a third type of human immunodeficiency virus hasimmediate epidemiological implications and consequences for blood banktesting. As has been shown, antibodies from people infected with thisvirus react preferentially with this virus, although these antibodiesalso crossreact with HIV-1 proteins. While it was possible to detect apositive reaction of ANT 70 NA serum in enzyme immunoassays,immunofluorescence assays and Western Blot assays based on HIV-1proteins, the fact that the positive signal was due to a crossreactioninevitably implies that the sensitivity of such tests will be less forantibodies produced in response to this virus. This was amplydemonstrated by the enzyme immunoassay results (FIG. 11). Furthermore,one criterion for seropositivity in the Western blot assay is thepresence of detectable antibodies to both a gag and/or pol protein andone of the envelope proteins. Since in the case of the two individualsinfected with ANT 70 and ANT 70 NA, respectively, crossreaction wasobserved to both HIV-1 p24 and the envelope proteins, the conclusionwhich is invariably drawn is that these individuals are infected withHIV-1 but for some reason fail to develop high titers against HIV-1. Itis possible therefore, that this virus is more widespread than iscurrently realized. From an epidemiological standpoint, it is essentialto develop specific diagnostic tests for this virus in order to evaluatethe limits of the geographical area in Africa in which the virus can befound, and to evaluate the extent to which this virus has beendisseminated.

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We claim:
 1. A process for making a monoclonal antibody that recognizes a protein or glycoprotein antigen of HIV-3 retrovirus (also known as HIV-1 subtype O virus), said retrovirus having the morphological and immunological characteristics of any of the retroviruses deposited in the European Collection of Animal Cell Cultures under V88060301, the process comprising:(a) infecting an animal with at least one antigen selected from a protein or a glycoprotein of HIV-3 to form antibody-producing cells; (b) fusing said antibody-producing cells with immortalized cells under hybridoma-forming conditions to form hybridomas; (c) selecting at least one hybridoma which secretes monoclonal antibodies which recognize the antigen as determined by western blot or by ELISA; and, (d) collecting monoclonal antibodies secreted by the hybridoma.
 2. The process of claim 1, wherein the antigen is a protein lysate of HIV-3.
 3. The process of claim 1, wherein the antigen is an internal core protein of HIV-3 selected from the group consisting of p12, p16, p25, and mixtures thereof.
 4. The process of claim 1, wherein the antigen is an envelope glycoprotein of HIV-3 selected from the group consisting of gp41, gp120, and mixtures thereof.
 5. A process for making a monoclonal antibody that recognizes a protein or glycoprotein antigen of HIV-3 retrovirus (also known as HIV-1 subtype O virus), said retrovirus having the morphological and immunological characteristics of any of the retroviruses deposited in the European Collection of Animal Cell Cultures under V88060301, the process comprising:(a) immortalizing B-cells derived from persons infected with HIV-3, at least one said B-cell secreting monoclonal antibodies which recognize a protein or glycoprotein of HIV-3 as determined by western blot or by ELISA; and, (b) collecting monoclonal antibodies secreted by the B-cell.
 6. The process of claim 5, wherein the protein or the glycoprotein of HIV-3 is a protein lysate of HIV-3.
 7. The process of claim 5, wherein the protein or the glycoprotein of HIV-3 is an internal core protein of HIV-3 selected from the group consisting of p12, p16, and p25.
 8. The process of claim 5, wherein the protein or the glycoprotein of HIV-3 is an envelope glycoprotein of HIV-3 selected from the group consisting of gp41 and gp120.
 9. In a process for producing monoclonal antibodies, the improvement comprising preparing monoclonal antibodies against at least one antigen selected from a protein or a glycoprotein of HIV-3 retrovirus (also known as HIV-1 subtype O virus), said retrovirus having the morphological and immunological characteristics of any of the retroviruses deposited in the European Collection of Animal Cell Cultures under V88060301.
 10. The process of claim 9, wherein the antigen is a protein lysate of HIV-3.
 11. The process of claim 9, wherein the antigen is an internal core protein of HIV-3 selected from the group consisting of p12, p16, p25, and mixtures thereof.
 12. The process of claim 9, wherein the antigen is an envelope glycoprotein of HIV-3 selected from the group consisting of gp41, gp120, and mixtures thereof. 